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Author Notes:

Address correspondence to Alexander A. Kolykhalov, a.kolykhalov@emory.edu

Subjects:

Research Funding:

Partial funding for this project was provided by Georgia Research Alliance (GRA) grant GRA.VL16.C3.

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Microbiology
  • Pharmacology & Pharmacy
  • RdRp
  • ZIKV
  • Zika virus
  • nonradioactive assay
  • polymerase assay
  • polymerase inhibitor
  • HEPATITIS-C VIRUS
  • DENGUE VIRUS
  • NS5 METHYLTRANSFERASE
  • ENZYMATIC METHOD
  • NUCLEOTIDE
  • NUCLEOSIDE
  • DNA
  • IDENTIFICATION
  • TRANSMISSION
  • TRIPHOSPHATE

Analysis of Ribonucleotide 5 '-Triphosphate Analogs as Potential Inhibitors of Zika Virus RNA-Dependent RNA Polymerase by Using Nonradioactive Polymerase Assays

Tools:

Journal Title:

Antimicrobial Agents and Chemotherapy

Volume:

Volume 61, Number 3

Publisher:

Type of Work:

Article | Final Publisher PDF

Abstract:

Zika virus (ZIKV) is an emerging human pathogen that is spreading rapidly through the Americas and has been linked to the development of microcephaly and to a dramatically increased number of Guillain-Barré syndrome cases. Currently, no vaccine or therapeutic options for the prevention or treatment of ZIKV infections exist. In the study described in this report, we expressed, purified, and characterized full-length nonstructural protein 5 (NS5) and the NS5 polymerase domain (NS5pol) of ZIKV RNA-dependent RNA polymerase. Using purified NS5, we developed an in vitro nonradioactive primer extension assay employing a fluorescently labeled primertemplate pair. Both purified NS5 and NS5pol can carry out in vitro RNA-dependent RNA synthesis in this assay. Our results show that Mn 2+ is required for enzymatic activity, while Mg 2+ is not. We found that ZIKV NS5 can utilize single-stranded DNA but not double-stranded DNA as a template or a primer to synthesize RNA. The assay was used to compare the efficiency of incorporation of analog 5′-triphosphates by the ZIKV polymerase and to calculate their discrimination versus that of natural ribonucleotide triphosphates (rNTPs). The 50% inhibitory concentrations for analog rNTPs were determined in an alternative nonradioactive coupled-enzyme assay. We determined that, in general, 2′-C-methyl- and 2′-C-ethynyl-substituted analog 5′-triphosphates were efficiently incorporated by the ZIKV polymerase and were also efficient chain terminators. Derivatives of these molecules may serve as potential antiviral compounds to be developed to combat ZIKV infection. This report provides the first characterization of ZIKV polymerase and demonstrates the utility of in vitro polymerase assays in the identification of potential ZIKV inhibitors.

Copyright information:

© 2017 American Society for Microbiology. All Rights Reserved.

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